The US population at large, and particularly military personnel and first responders, are at risk of radiation exposure due to the explosion of a nuclear device, a nuclear reactor accident, and the threat of radiation terrorism. There is no radiation medical countermeasure (RCM) drug approved by the FDA that meets the criterion of a gastrointestinal (GI) radiomitigator ? an agent which mitigates the acute GI radiation syndrome (GI-ARS) when administered after the exposure. Ionizing radiation kills cells that are unable to repair their DNA, primarily via mitotic catastrophe and apoptotic cell death. Post-irradiation genotoxic stress and cell injury is an unsolved medical problem. A critical barrier to progress in development of RCM drugs is that a traditional human clinical trial is not an option. Therefore, FDA approval of a RCM is done under the Animal Rule that requires detailed understanding of its mechanism of action, demonstration of its safety, and efficacy in animal models, and its safety in humans. In this transitional research proposal, we propose studies to fully satisfy the mechanism of action requirement of the Animal rule for Radioprotectin-1 (RP-1) a new radiation mitigator we developed with previous NIAID funding and develop a single-dose extended release formulation that meets CONPOS requirements of a RCM. Our overall goal is to prepare RP-1 for regulatory approval as a first-in-class synthetic GI radiation mitigator. RP-1 is the first specific agonist of the lysophosphatidic acid (LPA) receptor subtype 2 (LPA2) with picomolar EC50, which reduces radiation injury-induced mortality in mice. Our central hypothesis is that RP-1?activated, uniquely long-lasting (> 16h) signaling mediated by the LPA2 G protein-coupled receptor (GPCR), is responsible for mitigation of genotoxic stress and promotion of cell survival. Our hypothesis predicts that RP-1 achieves this via 1) obligate stimulation of the LPA2 GPCR, 2) sequential recruitment of supramolecular signaling interactomes responsible for the long duration of its action, 3) augmentation of DNA repair and 4) enhanced survival of LGR5 intestinal stem cells (ISC). Our objectives are: 1) determine in detail the unique molecular mechanism of how RP-1 acts via LPA2 to recruit the interactomes required for overcoming genotoxic stress, and 2) identify the specific subpopulation of ISC that is protected by RP-1 in vivo using transgenic mice that express fluorescent protein in the LGR5 marker bearing ISC and 3) develop a single dose extended release nanoparticle formulation that mitigates the GI-ARS. Although this information is necessary to move RP-1 forward toward regulatory approval, the body of knowledge we will generate also represents significant and previously unknown information concerning radioprotective signaling mechanisms. Our expected outcomes will include 1) establishing that LPA2-dependent recruitment of the IEX-1?TRIP6?ERK1/2-AKT interactome is required for mitigation of genotoxic stress; 2) defining the role of RP-1 effects that enhance DNA-dependent Protein Kinase- dependent DNA repair and prosurvival signals; 3) demonstrating that RP-1 is an effective mitigator of GI-ARS by protecting ISC in mice; 4) a single-dose extended-release RP-1 formulation. The impact of our project will directly affect our first-response options in treating patients with radiation injury. The aims of the project are: Aim 1. Test the hypothesis that RP-1 via LPA2 mediates long-lasting activation of DNA repair and pro- survival signaling in LGR-5 positive intestinal stem cells. Aim 2. Develop a nanoparticle-based extended-release RP-1 formulation for the treatment of the GI-ARS of mice and Rhesus macaques. All data obtained will be used for a RP-1 Drug Master file and presented to the FDA during our PRE-IND meeting in the final year of the project and shared with the research community at large.